Vaccine for neonatal calf diarrhea

ABSTRACT

A vaccine is prepared by passaging a coronavirus-like agent in tissue culture or by inactivating the viral agent. The vaccine is useful for immunizing bovine animals against calf diarrhea.

United States Patent [1 1 Mebus [451 Mar. 25, 1975 VACCINE FOR NEONATALCALF DIARRHEA [75] Inventor: Charles A. Mebus, Lincoln, Nebr.

[73] Assignee: The Board of Regents of the University of Nebraska,Lincoln, Nebr.

221 Filed: Oct. 12, 1973 211 Appl. No.: 405,997

Related US. Application Data [63] Continuation-impart of Ser. No.302,179, Oct. 30,

1972, abandoned.

[52] US. Cl ..195/1.3, 195/1.1, 424/89 [51] Int. Cl. C12k 5/00, C12k7/00 [58] Field of Search 195/1. 3; 424/89 [56] References Cited OTHERPUBLICATIONS Mebus et a1., Vet. Path. 11:375 (1973'). Mebus et al.,Amer. J. Vet. Res. 341145-150 (1973). Stair et al., Amer. J. Vet. Res.33: 1147-1155 (1972).

Mebus et al.,'Univ. Nebraska Coll. Agric. Home Econ.

Res. 31 1 1 1 233, Mar. 1969.

Primary Examiner-Shep K. Rose Attorney, Agent, or Firm-Alan D. Lourie;William H.

Edgerton [57] ABSTRACT A vaccine is prepared by passaging acoronavirus-like agent in tissue culture or by inactivating the viralagent. The vaccine is useful for immunizing bovine animals against calfdiarrhea.

4 Claims, N0 Drawings VACCINE FOR NEONATAL CALF DIARRHEA Thisapplication is a continuation-in-part of copending Ser. No. 302,179,filed Oct. 30, 1972 now abandoned.

The present invention relates to a vaccine against a newcoronavirus-like agent which is a causative factor in neonatal calfdiarrhea and to a method of obtaining said vaccine. 1n particular, theinvention consists of a method of attenuating the coronavirus-like virusby serial passage in fetal bovine kidney cells and to the vaccineobtained thereby. ln addition, a vaccine is prepared by inactivating thecoronavirus-like agent after it is propagated on cell cultures.

BACKGROUND AND IDENTIFICATION OF THE CORONAVlRUS-LIKE AGENT Theetiologic factors of neonatal calf diarrhea (NCD) are complex. Numerousorganisms have been implicated as causes for this disease, includingbacteria, fungi, mycoplasma, chlamydia, and viruses. Virusesspecifically enumerated have been bovine viral diarrhea, infectiousbovine rhinotracheitis, adenovirus, enterovirus, and HADEN virus. Otherviruses that have been reported as etiologic agents of NCD are calfpneumonia enteritis virus and a reovirus-like agent referred to as NCDvirus. The method of preparing a vaccine effective against this NCDreovirus-like agent is disclosed and claimed in application Ser. No.197,520, filed Nov.

In the course of field trials of the vaccine prepared from thereovirus-like agent, it was observed that in certain herds in westernNebraska inadequate control of the calf disease resulted. [Vet.Med/Small Anim. Clin., 67(2), 173 (1972)]. Typically, the calveshadreceived the vaccine during the first 24 hours of life, yet

diarrheas developed in several herds when the calves were 5 to days old.Diarrheal feces from these calves were examined for the reovirus-likevirus using the fluorescent antibody technique and were foundnegative.

Diarrheal feces from one of the vaccinated herds in which 90 per cent ofthe calves were affected when they were 7-12 days old were inoculatedvia duodenal injection into a Caesarean derived colostrum-deprived calf.This calf developed diarrhea from which bacteriafree fecal filtrateswere prepared and inoculated orally into gnotobiotic calves. Thesecalves developed diarrhea, thereby indicating another viral agent as thecause of diarrhea. Electron microscopic examination of diarrheal fecesfrom the experimental calves revealed a coronavirus-like agent. Similarviral particles were found in diarrheal feces both in vaccinated herdsparticipating in the field trials and in herds in which no vaccine hadbeen used.

This new coronavirus-like agent has now been purified and identified ingreater detail. We have discovered that this corona-like virus can bepropagated and modified on cell cultures into an effective vaccine. Wehave also discovered that an effective vaccine can be prepared byinactivating the virus after it has been grown on cell cultures.Therefore, our invention consists of vaccines effective against this newcoronaviruslike agent and of methods of preparing and administering thisvaccine. Purification and description of this agent have now beendescribed in Am. J. Vet. Res. 33, 1147-56 (1972).

Purification of the Coronavirus-like Agent Samples of fecal materialwere collected from diarrheal calves of 19 ranch herds in westernNebraska. Calves from 12 of the 19 herds had been previously inoculatedorally with NCD reovirus-like virus vaccine while calves from theremaining herds were unvaccinated. Fecal smears from diarrheal calveswere stained with an immunofluorescent conjugate to test for NCD virus(Univ. of Nebraska, Agri. Exper. Sta. Res. Bull. 233, 1969) and onlysamples negative for NCD virus were used. Diarrheal fecal materialcollected from naturally infected calves in the field and fromexperimentally infected calves was frozen immediately and stored at 20to 60C until viral purification was begun.

Sucrose density gradients were prepared and allowed to stand overnightat 4C in order to form a linear gradient before use. Gradients consistedof 8 ml each of 400, 300, 200 and mg of sucrose per milliliter ofdistilled deionized water. Decreasing concentrations were layered into2.5 by 8.9 cm cellulose nitrate centrifuge tubes.

All procedures leading to viral purification were conducted at 4C. Aclinical centrifuge and an ultracentrifuge were used.

The diarrheal fecal material was mixed with three volumes of deionizedwater and centrifuged at 5,000 g for 30 minutes. The supernate was thencentrifuged for 3 hours at 25,000 rpm in a type 30 rotor. The resultantpellet was dispersed by sonification for 30 seconds in 5% (w/v) sucrosesolution. The suspension will be referred to as the crude viralsuspension.

Crude viral suspension (5 ml) was layered on the gradients in the tubesand centrifuged at 25,000 rpm for 2 hours in a SW 27 rotor. Aftercentrifugation, bands were located by light scattering. Material fromeach band was collected from the top of the centrifuge tube using aPasteur pipet fitted with a propipette. Samples were dialyzed against 1%solution of ammonium acetate before electron microscopy was done. Thefraction containing virus (semipurified virus from the band that was 6cm from the meniscus) was submitted to further purification by sucrosedensity gradient centrifugation or by cesium chloride (CsCl) gradientcentrifugation.

The semipurified virus preparation was purified further by repeating thesucrose density gradient centrifugation process described above. Againbands were located and the band at 6.0 cm from the meniscus wascollected and dialyzed against 1% ammonium acetate to yield a purifiedvirus.

lsopycnic gradient centrifugations of the semipurified virus preparationwith CsCl were conducted by starting with a 30% solution (15 Gm of CsClplus 35 ml of deionized water). This solution (4 ml) was placed incellulose nitrate centrifuge tubes, and each tube was overlayed with 1.4ml of viral suspension. A SW 65 rotor was used at 60,000 rpm for 18hours, at which time apparent equilibrium had been reached. Therefractive index of the CsCl in the zone where the virus was located wasdetermined using a TS meter. The band at 2.9 cm from the meniscus wascollected and dialyzed against 1% ammonium acetate solution to give apurified virus.

The aforementioned crude viral suspension (15 ml) was also fractionatedby gel filtration on a 4.2 by 42 cm gel column (Sepharose 2B, PharmaciaFine Chemicals,

Inc, Piscataway, N.J.). The column had been previously equilibrated with0.9% NaCl and was developed with this salt solution. A flow rate of 6.0ml/hour was used. Fractions of 5 ml were collected, and the absorbancewas determined at 260 pm with a spectrophotometer. Materials from eachof the different peaks were combined and separately concentrated byultrafiltration.

A simplified procedure was also used for preparation of virus fromdiarrhea] fecal material from experimental calves. The fecal materialwas centrifuged directly at 3,400 g for 30 minutes. A portion of thesupernatant material was extracted twice with dichlorodifluoromethane.Aqueous phase (5 ml) and untreated supernatant (5 ml) material weresubjected to sucrose density gradient centrifugation as previouslydescribed.

The virus concentrated by density gradient centrifugation was used forelectron microscopic studies and also as an antigen for rabbitimmunization to prepare a conjugate for immunofluorescent analysis.White domestic rabbits about 6 months old were bled from the heart (14ml) and then injected intramuscularly at four sites with.0.25 ml persite ofa mixture of 0.4 ml antigen and 0.6 ml complete Freunds adjuvant.Five weeks after inoculation 70 ml of blood were collected by cardiacpuncture. Fluorescein labeled gamma globulin was prepared from the postinoculation serum by known methods (Proc. US. Livestock San. Assn., Oct.1968, 139-144).

The presence of the coronavirus-like agent could be detected in infectedintestine by the immunofluorescent technique using the conjugateprepared above. The specificity of this conjugate for thecoronavirus-like agent was clearly shown in the following series oftests. Sections of intestine from diarrheic calves which had beeninfected with coronavirus-like agent were stained with the conjugate andfluorescence of the villous cells was observed. lntestine from the samecalf had no fluorescence when stained with the conjugate for thereovirus-like agent referred to above. Sections of intestine fromdiarrheic calves which had been infected with the reovirus-like viruswere positive when stained with conjugate for the reovirus-like agentand negative when stained with the conjugate for the coronavirus-likeagent. Sections of a normal gnotobiotic calf intestine had nofluorescence when stained with the conjugate for the coronavirus-likeagent. Sections of a normal gnotobiotic calf intestine had nofluorescence when stained with the conjugate for the coronavirus-likeagent.

Morphology of the Virus The virus-containing band resulting fromgradient density ultracentrifugation purification was examined byelectron microscopic examination. Droplets from the bands were appliedto 200 mesh copper grids which had previously been coated with collodionand reinforced with evaporated carbon. The droplets were allowed toremain on the grids for 3 to minutes depending on the degree of lightscattering of the band that was being studied. Excess fluid was blottedoff with filter paper and then a droplet ofvanadatomolybdatephosphotungstic acid solution was applied to the grid.The stain was allowed to remain on the grid for l to 1.5 minutes beforethe excess was removed by using filter paper as a blotter.

The samples were examined with an electron microscope with an instrumentmagnification of 32,000 or greater. Size determinations were made bydimensional analysis of electron micrographs of the specimens anddiffraction grating replicas which were taken immediately after thespecimen electron micrographs without altering the settings of theinstrument.

Size of complete viral particles ranged from 107 to 160 ,um in the samemicrograph and had surface projections similar to those observed for thecoronaviruses. Average size of particles, including surface projections,as determined 'by electron microscopy, was 126 am. The nucleocapsid waspolymorphic and varied in size and shape from round to oblong. Envelopesof nucleocapsids were particularly obvious when the fringe was lost.Fringe width was variable to a maximum of 23 um. Surface projections ofwell-preserved particles were petal shaped, attached to the particle bya slender stalk, and an average of 11 pm in length.

The coronavirus-like agent described in this invention is easilydifferentiated on the basis of size and morphological features fromother viruses that have been proved or proposed to be an etiologicfactor in neonatal calf diarrhea. The coronavirus-like agent hereindescribed has different morphologic features and is larger thanenteroviruses, the virus of calf-pneumonia enteritis, bovine viraldiarrhea, neonatal calf diarrhea virus (reo-like), and HADEN virus. Thisnew agent is smaller and lacks the typical morphological features ofinfectious bovine rhinotracheitis virus. The buoyant density of purifiedcoronavirus-like agent was 1.24 using CsCl while the reovirus-like agenthad a buoyant density of 1.359 [Can. J. Comp. Med., 35 (1971)].

The physiological effects of the coronavirus-like agent also differ fromthat ofthe reo-like virus. The in cubation period following oralinoculation of gnotobiotic calves with the reovirus-like agent can be asshort as 13.5 to 14 hours while the shortest incubation period observedwith the coronavirus-like agent was 18 hours. In addition, reo-virusinfected gnotobiotic calves have diarrhea for 5 to 8 hours and thenappear normal 24 hours after the onset of diarrhea. On the other hand,coronavirus-like agent infected gnotobiotic calves develop diarrhea andmay continue to have diarrhea 5 or more days or may die 23 days afterdiarrhea begins.

PREPARATION AND USE OF THE VACCINE The following description illustratesmethods useful in carrying out our invention; however, it is to beunderstood that these are not limitative.

The coronavirus-like agent obtained from infected calves and describedherein was propagated on fetal bovine kidney cells. The virus may alsobe propagated on cells from other tissues or cell lines of bovine orother origin. Monolayer cell cultures are prepared by known methods andinoculated with the coronaviruslike agent. In general, monolayercultures are washed with Hanks balanced salt solution and then a viralinoculum is added and allowed to be absorbed for several hours,generally 2 hours, at about 37. A maintenance medium of Earles balancedsalt solution containing 0.5% lactalbumin hydrolysate (LAH), 0.1% yeastextract, and units penicillin and 200 1.4g streptomycin per milliliteris added and the culture is incubated at 30-40, preferably about 37, for210 days. The virus is harvested, e.g. by pouring off the supernatantfluid from the cells. The viral inoculum may be feces collected fromcoronavirus-infected calves or culture fluids from coronavirus-likevirus cultures. The feces can be used as collected or can be diluted inphosphate buffered saline (pH 7.2 centrifuged at 1,000 g for 20 minutesand the supernatant fluid used as the inoculum.

Other maintenance media may be used, for example, modified Eagle'smedium containing lactalbumin hydrolyzate. The choice of the medium iswithin the normal skill of the art.

An effective attenuated corona-like virus vaccine is obtained bypassaging purified virus in fetal bovine kidney cells a sufficicntnumber of times so that when an inoculated calf is challenged withvirulent virus, no disease is produced. One will generally need topassage the virus from 5 to 60 times, the passages being in primary orsecondary (primary cells which have been passaged once) cells or higherpassaged cells or cell lines. Higher passaged cells which have beenpassaged more than times may be considered cell lines. A preferredmethod involves a combination of passages on primary or secondary cellsand higher passaged cells or cell lines. Virus titers are determined bystandard methods. For example, cells inoculated with virus dilutions areincubated for 5 days in tubes containing fetal bovine kidney cells. Thepresence of virus is determined, e.g. by cytopathologic effects,immunofluorescence, or hemadsorption.

More specifically an effective attenuated vaccine was produced by thefollowing procedure. A bovine fetus was obtained and a large quantity ofprimary kidney cells was prepared. Several bottles and coverslipcultures were prepared from the primary cells and the remainder of theprimary cells were frozen using Earles balanced salt solution containing0.5% LAl-l, 10% adult bovine serum, and 10% dimethyl sulfoxide. Thebottle and coverslip cultures were inoculated with viruscontainingsolution as described herein and incubated at 37C. When a fewfluorescent cells were observed on the coverslip cultures some frozenprimary cells from the same fetus were thawed and propagated intomonolayer cultures by standard methods. Fluid from the infected primarycell culture was passed onto these secondary cell cultures. Afterincubation of 6-7 days, fluid from the secondary cell culture was usedto inoculate additional secondary cell cultures. From the 1st throughthe th passage, 2 ml of the fluid was used to inoculate an 8 ouncebottle as described heretofore. From the 15th to 25th passage, 2 ml ofthe fluid was added to the maintenance medium in the bottle. After the25th passage, a 1 ml inoculum is added to the maintenance medium in eachbottle. When the coronaviruslike agent had reached the 5th to th tissueculture passage, a series of consecutive passages of cells fromsecondary cells was initiated. At each passage of the virus only part ofthe cells were infected and the other uninfected cells were subculturedto become the next higher passage level of cells. This type of cellculture is referred to as the passaged cell culture. By continuing theconsecutive subcultural passages of the virus on passaged cell culture,a compatible cell-virus system was evolved. After 5-20 passages of thevirus in secondary cell cultures the virus was passaged 5-40 or moretimes in passaged cell cultures to produce an effective attenuatedcoronavirus-like virus vaccine. The period of incubation for the viralpassages decreases with increasing numbers of passages and can be from 2to 7 days for passages on higher passaged cells or cell lines. Eachpassage is conducted until a cytopathogenic effeet is obtained. Thevaccine may be produced on a fetal bovine kidney cell line or from othertissues or cell lines of bovine or other origin, for example, bovinelung, a bovine turbinate cell line, or procine kidney cells or celllines, by growing it in the tissue culture at about 37 until a suitablevirus titer is obtained and then harvesting the virus. Titers of 10 to10, preferably 10 to 10 are obtained. The vaccine may be used in theliquid state or lyophilized. It can be reconstituted with sterilediluent such as distilled water for later use.

The preferred product consists of an attenuated virus which has beenpassaged 12 times on secondary cells and 15 times on cells whichthemselves have been passaged nine to 27 times. The total passage levelwas thus 27. Vaccines of up to 35 passages have been prepared.

In addition, an effective inactivated vaccine is prepared byinactivating the coronavirus-like agent. In general, this is prepared byfirst propagating the agent on fetal bovine kidney cells as describedabove until an adequate titer is obtained. The titer should be as highas possible within the range stated above. Any cell passage level ofvirus, even an unattenuated virus, can be used. The virus is theninactivated by treating it at 20-40 with an inactivating agent known inthe art, such as formalin, B-propiolactone, ultraviolet radiation, orheat, for such a length of time and concentration of inactivating agentas to effectively inactivate the virus. These details are well known tothe art. An adjuvant may be added to enhance the antigenicity. The ad-30 juvant may be any of those known in the art including aluminumhydroxide gel, potassium alum, alginate, or an oil base or otheradjuvant such as mineral or other organic oil.

A typical procedure to prepare an inactivated vaccine is as follows. Thecoronavirus-like agent at the 27th passage level as described above isused. Formalin is added to the virus-containing culture fluids until0.2% concentration is obtained. These fluids are then allowed to standfor 1 to 2 days at 37. To the inactivated virus is added aluminumhydroxide gel adjuvant to a concentration of 10%. The pH is adjusted toabout 7.2 with aluminum hydroxide.

The attenuated vaccine can be administered orally in doses of l to 5 mlcontaining 10 to 10 TClD/ml to calves, preferably newborn, to induceimmunity to virulent virus. The inactivated vaccine is administeredparenterally in doses of 1 to 5 ml to pregnant cows 30-90 days prior tocalving or to calves at an early age as soon after birth as possible, toprovide protection from the coronavirus-like agent. I

In addition, a combination vaccine can comprise coronavirus and anyother viral agent, preferably inactivated, used to immunize calves orcows. A preferred combination includes inactivated coronavirus andreovirus, both of which are prepared by inactivating a live virus,either unattenuated or attenuated, by chemicals, radiation, or heat.Coronavirus can also be combined with bovine adenovirus, bovine virusdiarrhea, or other bovine pathogen such as E. coli or clostridia sp.

A combination vaccine containing coronavirus and reovirus is prepared bymixing equal amounts of both inactivated agents. One then adds formalinto a concentration of 0.2% and aluminum hydroxide gel to 10% by volume.The dosage of this vaccine is l to 5 ml administered parenterally to thepregnant cow. The combinations can be given either to calves or pregnantcows. The combination vaccines provide protection for the newborn calf,via colostral and/or milk antibodies. In addition, protection againstboth agents can be developed by immunizing the pregnant cow with theinactivated reovirus-like vaccine and then administering the attenuatedcoronavirus-like vaccine to the calf. Protection against both agents canalso be obtained by immunizing the pregnant cow with the inactivatedcoronavirus-like vaccine and then administering the attenuatedreovirus-like vaccine to the calf. Finally, a combination vaccinecomprising attenuated coronavirus and reovirus-like agents can be givento the pregnant cow.

The attenuated virus vaccine was tested for effectiveness byadministering the vaccine produced at various cell culture passagelevels to gnotobiotic calves at the age of about 7 hours. At a latertime the calves were challenged by inoculation with virulent virus. Theresults of these tests are summarized in Table l.

Calf l inoculated orally with 10 ml of cell culture fluid from the 14thpassage of the virus on secondary 20 fetal bovine kidney cells developeddiarrhea and recovered. At 9 days of age, the calf was inoculated orallyveloped mild diarrhea. Calf 3 was killed shortly after 'the onset ofdiarrhea; sections of the small intestine and colon stained withconjugate for the coronavirus-like agent had immunofluorescence in theepithelium of the intestinal villi and colon. Calf4 inoculated withvirulent virus in diarrheal feces when 6 days old remained normal. Calf6 inoculated orally with 5 ml of 19th passage virus grown on secondaryfetal bovine kidney cells developed diarrhea. Twelve calves (No. 7through 13 and 15 through 19) inoculated orally with 5 ml of 13th to25th passage virus of which at least 5 passages were on higher passagesof fetal bovine cells did not develop diarrhea and, except for calf 18,remained normal after oral inoculation with feces containing virulentvirus.

A combination vaccine containing inactivated coronavirus and reoviruswas evaluated in adult calves from 2. ranches. The vaccine was preparedfrom 27 passage coronavirus, inactivated with formalin, and 201 passage(primary bovine kidney and bovine kidney cell line), reovirus,inactivated with formalin. The inactivated viruses were mixed togetherin equalamounts and aluminum hydroxide gel was added to 10%.

TAB LE 1 Result of Challenge Age of Calf Calf No. No. of Virus PassagesResult of Virus Inoculation Inoculation (Days) 1 14 O D N 9 2 l5 N N 3 3l6 0 d killed 4 16 0 d N 6 5 Control for challenge virus D 3 6 l9 0 Dnot challenged 7 5 8 N N 4 8 6 7 N N 3 9 9 5 N N 3 l0 8 9 N N 3 l l 9 7N N 3 12 l l 8 N N 4 13 l l 9 N N 5 14 Control for challenge virus D 3l5 ll 11 N N 4 l6 l0 l l N N 3 17 12 N N 3 l8 10 15 N d 4 l9 10 13 N N 4D diarrhea; N normal Passages on primary or secondary cells and passageson passaged cells TABLE 2 No. of Cows No. of calves treated for scourstreated Ranch No. l 242 vaccinated twice 3 1% 85 unvaccinated 77 91%Ranch No. 2 (a) 600 vaccinated twice (400 calved in shed l04 26% wherebacterial infection was found; 200 calved outside l0 5% of shed) (b) 350vaccinated once 17 5% I300 unvaccinated 195 15% (c) 750 vaccinated twice7 l% (d) 250 vaccinated twice 3 1% 250 unvaccinated 10% with 10 ml ofdiarrheal feces (virulent virus) in 20 ml PBS and remained normal. Calf2, inoculated with 10 ml of 15th passage of virus on secondary fetalbovine kidney did not develop diarrhea and remained normal after it wasgiven challenge inoculum of virulent virus.

Calves 3 and 4 inoculated with 10 ml of 16th passage virus grown onsecondary fetal bovine kidney cells de- We claim:

1. A process for attenuating a coronavirus-like calf diarrhea viruscomprising passaging said virus from five to about times in tissueculture which supports the growth of the virus until the virus, wheninoculated into newborn calves, prevents the development of disease uponsubsequent challenge with virulent virus.

4. A process for preparing a further quantity of attenuatedcoronavirus-like calf diarrhea virus comprising further growth of anattenuated coronavirus-like calf diarrhea virus in fetal bovine kidneycell culture, said further growth being for a length of time sufficientto permit growth of a larger amount of said virus and then harvestingthe resultant viral material.

1. A PROCESS FOR ATTENUATING A CORONATIRUS-LIKE CALF DIARRHEA VIRUSCOMPRISING PASSAGING SAID VIRUS FROM FIVE TO ABOUT 6O TIMES IN TISSUECULTURE WHICH SUPPORTS THE GROWTH OF THE VIRUS UNTIL THE VIRUS, WHENINOCULATED INTO NEWBORN CALVES, PREVENTS THE DEVELOPMENT OF DISEASE UPONSUBSEQUENT CHALLENGE WITH VIRULENT VIRUS.
 2. The process of claim 1 inwhich the passages are in a tissue culture of fetal bovine kidney cellsat 30*-40*.
 3. The process of claim 2 in which the total passages are10-40 of which 5-10 passages are in primary or secondary fetal bovinekidney cell cultures and the remainder are in higher passaged fetalbovine kidney cell cultures.
 4. A process for preparing a furtherquantity of attenuated coronavirus-like calf diarrhea virus comprisingfurther growth of an attenuated coronavirus-like calf diarrhea virus infetal bovine kidney cell culture, said further growth being for a lengthof time sufficient to permit growth of a larger amount of said virus andthen harvesting the resultant viral material.